Ex Vivo Expansion of Human Hematopoietic Stem Cells

ABSTRACT

Methods and kits for expanding the number of hematopoietic stem cells are provided. The methods comprise incubating cells in medium comprising isolated IGFBP-2 and an angiopoietin-like protein (Angptl). Expanded HSCs are provided as well as culture media and kits for the expansion of human HSCs in a defined medium. Methods of administering expanded human HSCs to and individual are provided as well as methods of treating an individual by administering certain growth factors and cytokines.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/598,770 filed Nov. 4, 2009, which is a national stage application ofInternational Application No. PCT/US2008/062365 filed May 2, 2008, whichclaims the benefit of and priority to U.S. Provisional Application No.60/927,668 entitled “Ex Vivo Expansion of Human Hematopoietic StemCells” filed May 4, 2007, now expired and U.S. Provisional ApplicationNo. 61/014,006 entitled “Method for Expansion and Analysis of CulturedHematopoietic Stem Cells” filed Dec. 14, 2007, now expired, the contentsof all of which applications are herein incorporated by reference intheir entirety.

GOVERNMENT FUNDING

This technology was made with support from the United States governmentunder grant numbers RO1 DK 067356-01, 1 K01 CA 120099-01, and075/P-IRFT, awarded by the National Institute of Health, and the UnitedStates government has certain rights in the technology.

FIELD

This disclosure relates to methods and compositions for the in vitroand/or ex vivo propagation of human hematopoietic stem cells.

BACKGROUND OF THE TECHNOLOGY

The hematopoietic stem cell (HSC), through proliferation anddifferentiation, gives rise to all lymphoid, myeloid, and erythroidcells. Pluripotent HSCs are thus the basis of bone marrowtransplantation and are considered attractive target cells forhematopoietic gene therapy for many clinical conditions. However, theseimportant clinical applications have been severely hampered by the lownumbers of HSCs that can be obtained from an animal, as well asdifficulties in culturing HSCs in vitro and expanding HSCs forsubsequent administration to a patient.

Culture and propagation of stem cells, such as hematopoietic stem cells,typically requires supplementation with unknown factors that allow thestem cells to survive and multiply in number. The unknown factors can besupplied by co-culturing the stem cells with feeder cells which secretean undefined panel of factors, or can be supplied by adding undefinedserum products to the growth medium. Such supplemented medium containsmany unknown factors and therefore is not chemically defined.

The presence of unknown factors is problematic when the stem cells arebeing prepared for in vivo use, especially in humans. In many instances,the unknown factors are from non-human sources (such as bovine serumproducts). The non-human components may cause an immune reaction in therecipient, or the undefined components could include undetectedpathogenic agents such as prions or viruses that would be detrimental tothe recipient of the stem cells. There is a need for methods andcompositions that allow the in vitro and/or ex vivo propagation of humanHSCs in a chemically defined medium, while maintaining the pluripotencyof the propagated cells.

SUMMARY

Provided herein is a defined culture medium for expanding humanhematopoietic stem cells. The defined medium includes certain growthfactors that synergize with each other to stimulate expansion of humanHSCs. Surprisingly, as demonstrated herein, a factor produced bynon-transfected 293T cells promotes the in vitro expansion of humanHSCs. That factor is shown herein to be insulin-like growth factorbinding protein 2 (IGFBP-2). The finding that IGFBP-2 promotes theexpansion of human hematopoietic stem cells is unexpected in light ofthe inhibitory effects that exogenous IGFBP-2 has on cell proliferationin different IGF-dependent cell culture systems. (Hoeflich, et al.,Canc. Res. 61:8601-8619 (2001)).

As demonstrated herein, IGFBP-2, in combination with one or more Angptlproteins promotes the expansion of human HSCs in a defined culturemedium. In some embodiments, human HSCs are expanded in a defined mediumby 250 fold or more.

In some embodiments, the method expanding human HSCs comprisesincubating human cells in a defined culture medium. The defined culturemedium can comprise IGFBP-2 and an angiopoietin-like protein (Angptl).In some embodiments, the defined culture medium can comprise IGFBP-2,Angptl5, fibroblast growth factor 1 (FGF-1), thrombopoietin (TPO), andstem cell factor (SCF). In some embodiments, the method comprisesincubating human cells for five days in a defined culture medium.

In some embodiments, the human cells are primary human cells. In someembodiments, the human cells include at least one cell that is capableof differentiating into one or more blood cell types. In someembodiments, the human cells include at least one hematopoietic stemcell.

In some embodiments, the human cells have been selected for cells thatexpress a surface marker selected from the group consisting of CD133 andCD34 prior to being incubated.

Methods of administering hematopoietic stem cells to an individual arealso provided. In some embodiments, the method comprises obtaining cellsfrom the individual or a donor. In some embodiments, at least one of thecells is capable of differentiating into one or more blood cell types.The cells are expanded in vitro as provided herein. In some embodiments,the cells are incubated in a defined culture medium comprising anIGFBP-2 and a growth factor selected from the group consisting ofangiopoietin 2 or an Angptl. The incubated cells are then administeredinto the individual.

Methods of treating a patient comprising administering an IGFBP-2 and anAngptl to the individual are provided.

Hematopoietic stem cells that have been expanded in vitro as describedherein are also provided.

Culture media and kits for expanding human hematopoietic stem cells invitro are also provided. In some embodiments, the kit comprises adefined medium suitable for culturing hematopoietic stem cells, anisolated IGFBP-2, and another growth factor selected from the groupconsisting of angiopoietin 2 or an Angptl. The growth factors can besupplied as separate components, a cocktail, or can be supplied alreadyin combination with HSC growth medium.

In addition, methods are provided for expanding stem cells in culture,including hematopoietic stem cells, by culturing a population of cellsthat contains stem cells in a culture medium which contains an effectiveamount of an angiopoietin, such as angiopoietin 2, under conditionssufficient for expansion of the cells. Isolated hematopoietic stem cellsare also provided wherein the isolated hematopoietic cells specificallybind an angiopoietin. Culture media and kits for expanding hematopoieticstem cells in vitro are also provided. The culture media and kitscomprise an angiopoietin, such as angiopoietin 2 and instructions forexpanding hematopoietic stem cells in vitro.

As a result of the methods and compositions provided herein, human HSCscan be expanded in a defined medium while maintaining pluripotency. Thecells can be expanded in vitro for research use, or can be expanded invitro for subsequent administration to an individual (also referred toherein as ex vivo expansion).

The various embodiments described herein can be complimentary and can becombined or used together in a manner understood by the skilled personin view of the teachings contained herein.

BRIEF DESCRIPTION OF FIGURES

FIG. 1A shows total cell number versus days in culture of total humancord blood cells in the presence of Angptl5 (squares) or Angptl3(diamonds).

FIG. 1B shows the amount of human chimerism in the bone marrow ofNOD/SCID mice transplanted with 1×10⁶ uncultured human mononuclear cordblood cells (col. 1), or the progeny of 1×10⁶ initial human cord bloodcells cultured in serum free STIF plus Angptl5 (col. 2) or Angptl3 (col.3). Each symbol represents the engraftment of a single transplantedmouse assayed at two months post-transplant (n=5−12). (* Significantlydifferent from lane 1 value. Student's t-test, p<0.001.)

FIG. 2A is a bar graph showing percent repopulation using murine HSCsafter culturing in serum-free IMDM supplemented with 10 ng/ml SCF, 20ng/ml TPO, 20 ng/ml IGF-2, and 10 ng/ml FGF-1, bar 1; freshly collectedconditioned medium from 293T cells, bar 2; or in the same conditionedmedium after freeze/thaw, bar 3.

FIG. 2B, top panel shows a representative FACS analysis of therepopulation of myeloid and lymphoid lineages in mice that receivedcultured cells from conditions represented by bar 2 of FIG. 2A, at 5months post-transplant and a bar graph; the bottom panel shows a summaryof the percent repopulation data from six mice that received culturedcells from conditions represented by bar 2 of FIG. 2A for T-lymphoid(bar 1), B-lymphoid (bar 2), and myeloid (bar 3) cells.

FIG. 3 shows a Western blot of IGFBP-2 (lane 1), serum-free 3T3conditioned medium (lane 2), and serum-free 293T conditioned medium(lane 3) separated on an SDS PAGE gel and probed with anti-IGFBP-2antibody.

FIG. 4A shows percent repopulation 1 month (left panel) and 4 months(right panel) after engraftment of mice with murine HSCs after culturingin STIF medium plus Angptl3 (col. 1 and 4), STIF medium plus Angptl3 andIGFBP-2 (col. 2 and 5), STIF medium plus Angptl3 and Timp-1 (col. 3 and6).

FIG. 4B shows percent repopulation 1 month (left panel) and 4 months(right panel) after engraftment of mice with murine HSCs after culturingin STF medium plus Angptl3 (col. 1 and 4), STF medium plus IGFBP-2 (col.2 and 5), STF medium plus Angptl3 and IGFBP-2 (col. 3 and 6).

FIG. 4C shows limiting dilution analysis of the repopulating ability ofadult BM SP CD45⁺Sca-1⁺ cells before culture (left) and after culturefor 21 days in conditioned STF medium containing 100 ng/ml of purifiedAngptl3 and 500 ng/ml IGFBP-2 (right).

FIG. 5A shows cell number over time in days of human HSCs cultured inSTF medium containing Angptl 5 (squares), or cultured in STF medium(diamonds).

FIG. 5B shows % repopulation by 8000 fresh cells (col. 1), 15000 freshcells (col. 2), 8000 cells cultured in STF medium (col. 3), or 8000cells cultured in STF medium containing Angptl5 and IGFBP-2 (col. 4).

FIG. 5C shows FACS analysis of human hematopoietic engraftment at 2months in a representative mouse that was transplanted with uncultured(fresh) or cultured human cord blood CD133⁺ cells.

FIG. 5D shows the summary of multilineage engraftment data from micetransplanted with uncultured cells (left panel) and cells cultured inSTF medium containing Angptl5 and IGFBP-2 (right panel) showing %repopulation with myeloid (CD15/66b+, cols. 1, 4), B-lymphoid(CD34-CD19/20⁺, cols. 2, 5), and primitive (CD34⁺, cols. 3, 6) humancells.

FIG. 5E shows % repopulation of secondary recipients with totalhematopoietic (CD45/71⁺, col. 1), myeloid (CD15/66b⁺, col. 2),B-lymphoid (CD34-CD19/20⁺, col. 3), and primitive (CD34⁺, col. 5) humancells, transplanted with bone marrow from the primary mice transplantedwith cultured in STF medium containing Angptl5 and IGFBP-2 (lane 4 ofFIG. 5B) and transplanted into sublethally irradiated secondaryrecipients.

FIG. 6A shows total cell number over time of 2×10⁵ human cord bloodCD133⁺ cells in STF medium containing Angptl5 and IGFBP-2 cultured inlow levels of O₂ (diamonds) and normal levels of O₂ (squares).

FIG. 6B shows the number of CD34⁺ primitive cells over time for humanHSCs cultured in STF medium containing Angptl5 and IGFBP-2 cultured inlow levels of O₂ (diamonds) and normal levels of O₂ (squares).

FIG. 6C shows limiting dilution analysis of the repopulating ability ofcells before culture.

FIG. 6D shows limiting dilution analysis of the repopulating ability ofcells after culture for 10 days in STF medium containing 500 ng/ml ofAngptl5 and 100 ng/ml IGFBP-2 in low levels of O₂ (squares) and normallevels of O₂ (diamonds).

FIG. 6E shows multilineage engraftment in NOD/SCID recipientstransplanted with 20,000 uncultured CD133⁺ cells (left panel, n=8) orcultured progeny from 5,000 initial CD 133⁺ cells at normal O₂ (rightpanel, n=10).

FIG. 7 is a bar graph showing percent repopulation 4 months aftertransplant with 20 CD45.2 bone marrow SP Sca-1⁺ CD45⁺ cells cultured inSTIF medium with angiopoietin 2 together with 1×10⁵ CD45.1 bone marrowcells into CD45.1 recipients (n=4−5).

FIG. 8 shows SEQ ID NOs. 3-6, amino acid sequences for exemplaryangiopoietin-like proteins.

FIG. 9 shows SEQ ID NO. 1, the amino acid sequence for an exemplaryangiopoietin 2 protein and SEQ ID NO. 2, the amino acid sequence for anexemplary IGFBP-2 protein.

DETAILED DESCRIPTION

Methods for propagating and/or expanding hematopoietic stem cells (HSCs)are provided, as well as human HSCs produced by the methods. In someembodiments, the human HSCs are expanded in a defined medium. As aresult of the methods provided herein, ex vivo expanded human HSCs areavailable that are free from unknown factors or contaminants typicallypresent in cultured cells.

Culture Medium

As described herein, suitable cells are incubated in a defined (alsoreferred to herein as chemically defined) medium. Defined or chemicallydefined medium refers to a nutritive medium for culturing cells whereevery component and quantity thereof present in the medium is known. Insome embodiments, the medium is a liquid. In other embodiments, themedium can be a solid such as a tablet or a powder or semisolid materialsuch as a gel. In still other embodiments, the medium can be a liquidthat includes a solid structure such as a mesh, porous bead(s), and thelike. The defined medium can comprise a base mixture of components, suchas Dulbecco's MEM, IMDM, X-Vivo 15 (Cambrex), RPMI-1640 and StemSpan(Stem Cell Technologies). The base mixture can be supplemented withknown quantities of other components such as heparin, serum albumin,insulin, transferrin, and the like, or combinations thereof. In someembodiments, the medium is supplemented with 10 μg/ml heparin. The addedcomponents can be derived, for example, from any suitable animal source,including, human, bovine, and murine sources. For example, StemSpancomprises IMDM supplemented with bovine serum albumin, human insulin,and human transferrin. The added components and growth factors can beisolated from a biological source (such as tissue, serum, or conditionedmedium) or can be recombinantly produced. Suitable hosts for producingrecombinant growth factors or other components include, for example,bacteria, yeast, or cell culture. The cell culture can be, for example,insect cell culture, or mammalian cell culture. The growth factor can beglycosylated. In some embodiments, the growth factor is glycosylated inthe same or substantially the same manner as the naturally occurringgrowth factor. The growth factors or other added components describedherein can be from any suitable animal, including, for example, mouse,non-human primate, and human.

An “isolated” or “purified” component or growth factor is substantiallyfree of other materials with which it is associated with when produced(e.g., as produced by the biological source or by recombinant methodssuch as expression in transfected cells). In some embodiments, isolatedmeans less than 0.1%, less than 0.01%, or less than 0.001% of the othermaterials with which the component or growth factor is associated withwhen produced is present. In another embodiment, the defined medium isserum free.

The exemplary sequences of growth factors having the GenBank AccessionNos. provided herein are hereby incorporated by reference.

IGFBP-2

In some embodiments, the defined medium includes isolated insulin-likegrowth factor binding protein 2 (IGFBP-2). IGF Binding Proteins (IGFBPs)are a family of circulating proteins that bind IGF-1 and IGF-2 with anaffinity equal or greater than that of the IGF receptors. IGFBP-2 isalso known to have inhibitory effects on cell proliferation in differentIGF-dependent cell culture systems. (Hoeflich, et al., Canc. Res.61:8601-8619 (2001)). Surprisingly, as demonstrated herein, IGFBP-2 hasa positive effect on the in vitro expansion of human HSCs.

An exemplary IGFBP-2 protein sequence is provided, for example, inGenBank as Accession Number AAA36048 (human insulin-like growth factorbinding protein 2; SEQ ID NO: 2, FIG. 9). In addition to IGFBP-2, theskilled artisan will further appreciate that suitable IGFBP-2 includesthose proteins and/or polypeptides that have changes in the naturallyoccurring amino acid sequence wherein the altered sequence retains atleast some functional ability of native IGFBP-2. Suitable alterationsinclude changes to or elimination of non-essential amino acid residuesas well as conservative amino acid changes (e.g., replacing an aminoacid residue with an amino acid residue having a similar side chain).

Suitable IGFBP-2 shares at least 60% sequence identity with SEQ ID NO.9. In other embodiments, suitable IGFBP-2 shares at least 70% or atleast 80% or at least 90%, or at least 95%, or at least 96, 97, 98, or99% sequence identity with SEQ ID NO. 9 or a biologically active portionthereof.

Suitable analogs of IGFBP-2 include fragments retaining the desiredactivity and related molecules. Molecules capable of binding thecorresponding receptor of IGFBP-2 and initiating one or more biologicalactions associated with binding to the IGFBP-2 receptor are also withinthe scope of the technology (e.g., methods, HCSs, media, and kits)provided herein.

Angiopoietin-Like Proteins

The one or more angiopoietin-like protein (Angptl) can be any member ofa family of secreted glycosylated proteins that are similar in structureto angiopoietins (Oike et al., Int. J. Hematol. 80:21-8 (2004)). Angptlproteins contain an N-terminal coiled-coil domain and a C-terminalfibrinogen-like domain. Unlike angiopoietins, Angptl proteins do notbind to the tyrosine kinase receptor Tie2. Angptl proteins includeAngptl 1, 2, 3, 4, 5, 6, and 7. Angptl proteins also includemicrofibrillar-associated glycoprotein 4 (Mfap4), and analogs andequivalents thereof. Angptl2 has been described by Kim, I. et al. J BiolChem 274, 26523-8 (1999)). In addition, Angptl proteins are availablecommercially (R&D Systems, Abnova Corp). In one embodiment, the Angptlis Angptl 3. In another embodiment, the Angptl is Angptl 5.

Exemplary Angptl proteins are provided, for example in GenBank asAccession Number AAH12368 (human Angptl 1: SEQ ID NO 3; human Angptl2precursor; SEQ ID NO: 4) Accession Number AAH58287 (human Angptl3precursor; SEQ ID NO: 5) Accession Number AAH23647 (human Angptl4; SEQID NO: 6) and Accession Number AAH49170 (human Angptl5; SEQ ID NO: 7).SEQ ID NOs: 3 through 7 are shown in FIG. 8. Other suitable Angptlproteins share at least 60% sequence identity with any one of SEQ IDNOs: 3 to 7. In other embodiments, suitable Angptl proteins share atleast 70% or at least 80% or at least 90%, or at least 95%, or at least96, 97, 98, or 99% sequence identity with an exemplary Angptl sequencesuch as SEQ ID NOs: 3, 4, 5, 6, or 7, or biologically active portionsthereof. An exemplary sequence for Angptl7 is found in GenBank AccessionNo. AAH01881. An exemplary sequence for Mfap4 is found in GenBankAccession No. NP_(—)002395.

In addition to sequences provided above for Angptls, the skilled artisanwill further appreciate that suitable Angptls include those proteinsand/or polypeptides that have changes in the naturally occurring aminoacid sequence wherein the altered sequence retains at least somefunctional ability of the native Angptl. Suitable alterations includechanges to or elimination of non-essential amino acid residues as wellas conservative amino acid changes (e.g., replacing an amino acidresidue with an amino acid residue having a similar side chain).

Suitable analogs of Angptls include fragments retaining the desiredactivity and related molecules. For example, a suitable analog of anAngptl is a fragment of the angiopoietin-like protein containing thecoiled coil domain. For example, the coiled coil domain of anangiopoietin-like protein. Another analog is the fibrinogen-like domain.Fragments of Angptls such as the coiled-coil domain and thefibrinogen-like domain may be easier to express and to purify comparedto full-length protein. Molecules capable of binding the correspondingreceptor of the Angptl and initiating one or more biological actionsassociated with binding to the Angptl receptor are also within the scopeof the technology provided herein.

Angiopoietin 2

In some embodiments, the defined medium includes angiopoietin 2. Anexemplary angiopoietin 2 protein sequence is provided, for example, inGenBank as Accession Number NP_(—)001138 (human angiopoietin 2; SEQ IDNO: 1, FIG. 9). In addition, the skilled artisan will further appreciatethat suitable angiopoietin 2 includes proteins and/or polypeptides thathave changes in the naturally occurring amino acid sequence wherein thealtered sequence retains at lease some functional ability of nativeangiopoietin 2. Suitable alterations include changes to or eliminationof non-essential amino acid residues as well as conservative amino acidchanges (e.g., replacing an amino acid residue with an amino acidresidue having a similar side chain).

Suitable angiopoietin 2 shares at least 60% sequence identity with SEQID NO. 1. In other embodiments, suitable angiopoietin 2 shares at least70% or at least 80% or at least 90%, or at least 95%, or at least 96,97, 98, or 99% sequence identity with SEQ ID NO. 1 or a biologicallyactive portion thereof.

Suitable analogs of angiopoietin 2 include fragments retaining thedesired activity and related molecules. Molecules capable of binding thecorresponding receptor of angiopoietin 2 and initiating one or morebiological actions associated with binding to the angiopoietin 2receptor are also within the scope of the technology provided herein.

Other Growth Factors

In addition to IGFBP-2, angiopoietin 2, or one or more Angptls, othergrowth factors or cytokines useful to promote expansion of hematopoieticstem cells in methods of the technology can include one or more of:fibroblast growth factor (FGF), insulin growth factor, thrombopoietin(TPO), and stem cell factor (SCF). Accordingly, in another embodiment,the media includes at least two of FGF, IGF, TPO and SCF or analogs andequivalents thereof. Equivalents thereof include molecules havingsimilar biological activity to these factors (i.e. FGF, TPO, IGF andSCF) as wild-type or recombinantly produced cytokines. Analogs includefragments retaining the desired activity and related molecules. Forexample, TPO is a ligand of the mp1 receptor, thus molecules capable ofbinding the mp1 receptor and initiating one or more biological actionsassociated with TPO binding to mp1 are also within the scope of thetechnology. An example of a TPO mimetic is found in Cwirla et. al.,Science 276:1696 (1997).

Cytokines and growth factors are commercially available from severalvendors such as, for example, Amgen (Thousand Oaks, Calif.), R & DSystems (Minneapolis, Minn.) and Immunex (Seattle, Wash.).

As indicated above, the concentrations of cytokines or growth factorsrange from about 0.1 ng/mL to about 1.0 μg/mL. In another embodiment,from about 1 ng/mL to about 500 ng/mL of the factor is used. In anotherembodiment, from about 10 ng/ml to 100 ng/ml of the factor is used.Other useful concentrations of the growth factors can be readilydetermined by one of ordinary skill in the art using the teachingscontained herein.

In another embodiment, FGF-1, TPO, and SCF are also included in themedium. In another embodiment the SCF is present at 10 ng/ml, TPO at 20ng/ml, and FGF-1 at 10 ng/ml. In another embodiment, IGF-2, FGF-1, TPO,and SCF are also included in the medium. Other useful concentrations ofthe growth factors or cytokines can be readily determined by one ofordinary skill in the art using the teachings contained herein.

As described herein, identity or homology to a given amino acid sequencecan be determined as the percentage of identity between two sequences.The homology can be determined using methods known in the art, such asby means of computer programs such as GAP provided in the GCG programpackage (Program Manual for the Wisconsin Package, Version 8, August1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of MolecularBiology, 48, p. 443-453).

Ex Vivo Cultures of Hematopoietic Stem Cells Cells

The cell or cells to be cultured can include any cell that is capable ofdifferentiating into one or more blood cell types. Exemplary blood celltypes include phagocytic immune cells (e.g., granulocytes), monocytes(e.g., macrophage precursor cells), macrophages, eosiniphils,erythrocytes, platelet forming cells (e.g., megakaryocytes), Tlymphocytes, B lymphocytes, and natural killer (NK) cells. Suitablecells include primary cells obtained from an individual or donor.Suitable cells can also be capable of self renewal, that is, capable ofpropagating or increasing in number and remaining at the samedevelopmental stage as the parent cell.

Suitable cells can be isolated, for example, from any known source ofhematopoietic stem cells, including, but not limited to, bone marrow,peripheral blood, mobilized peripheral blood (MPB), fetal liver, andumbilical cord blood. Umbilical cord blood is discussed, for example, inIssaragrishi et al., N. Engl. J. Med. 332:367-369 (1995). Bone marrowcells can be obtained from a source of bone marrow, including but notlimited to, ilium (e.g., from the hip bone via the iliac crest), tibia,femora, vertebrate, or other bone cavities. Other sources of stem cellsinclude, but are not limited to, ES cells, embryonic yolk sac, fetalliver, and fetal spleen. Methods for obtaining cells from an individualor donor are well known in the art.

For isolation of bone marrow, an appropriate solution can be used toflush the bone, including, but not limited to, salt solution, optionallysupplemented with fetal calf serum (FCS) or other naturally occurringfactors, in conjunction with an acceptable buffer. In an embodiment, thebuffer is at low concentration, generally from about 5 to about 25 mM.Convenient buffers include, but are not limited to, HEPES, phosphatebuffers and lactate buffers. Bone marrow can also be aspirated from thebone in accordance with conventional techniques.

Suitable cells and the hematopoietic cells of the technology can bederived from any animal, where hematopoietic stem cells are present.Suitable animals include human, non-human primate, cow, horse, dog, cat,mouse and the like. In an embodiment, the cells are human cells, instill another embodiment, the cells are murine cells.

Animal models for long-term engrafting potential of candidate humanhematopoietic stem cell populations include the non-obesediabetic/severe combined immunodeficiency mouse (NOD/SCID) model, theSCID-hu bone model (Kyoizumi et al. (1992) Blood 79:1704; Murray et al.(1995) Blood 85(2) 368-378) and the in utero sheep model (Zanjani et al.(1992) J. Clin. Invest. 89:1179). For a review of animal models of humanhematopoiesis, see Srour et al. (1992) J. Hematother. 1:143-153 and thereferences cited therein. An in vitro model for stem cells is thelong-term culture-initiating cell (LTCIC) assay, based on a limitingdilution analysis of the number of clonogenic cells produced in astromal co-culture after 5 to 8 weeks (Sutherland et al. (1990) Proc.Nat'l Acad. Sci. 87:3584-3588). The LTCIC assay has been shown tocorrelate with another commonly used stem cell assay, the cobblestonearea forming cell (CAFC) assay, and with long-term engrafting potentialin vivo (Breems et al. (1994) Leukemia 8:1095).

As used herein, expansion or propagation includes any increase in cellnumber. Expansion includes, for example, an increase in the number ofhematopoietic stem cells over the number of HSCs present in the cellpopulation used to initiate the culture. The methods provided hereinprovide for the increased survival of existing cells, such ashematopoietic stem cells. The term survival refers to the ability tocontinue to remain alive or function.

The methods provided herein can be used to stimulate the expansion ofany stem cells which expand in the presence of angiopoietin 2, and/or anangiopoietin-like protein and/or IGFBPs, including other types of adultstem cells such as endothelial progenitor cells (Shi, Q. et al. (1998),Blood. 92, 362-367), bone marrow stromal stem cells (Owen, M. (1988), J.Cell Science Supp. 10, 63-76), mesenchymal stem cells (Pittenger, M. F.and Marshak, D. R. (2001), Marshak, D. R., Gardner, D. K., and Gottlieb,D. eds. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press349-374), and skeletal muscle stem cells (Gussoni, E., et al. (1999),Nature. 401, 390-394), embryonic stem cells, as well as others. Inanother embodiment, the stem cells are endothelial progenitor cells,which are believed to share the same precursor—hemangioblasts—as HSCs.

Subpopulations of cells can also be used in the methods provided herein.For example, a purified “side population” (SP) cells obtained from bonemarrow or other sources can be used. Other enriched populations of HSCscan also be used. Methods for isolating enriched populations of HSCs areknown to those in the art, e.g. methods for obtaining SP cells aredescribed in Goodell et al., J. Exp. Med. 183, 1797-806 (Apr. 1, 1996).

In addition, a subpopulation of cells enriched for stem cells can beused in the methods described herein. Separation of stem cells from acell population can be performed by any number of methods, includingcell sorting, (e.g., fluorescence activated cell sorting) magneticbeads, and packed columns. The methods typically rely on the presence ofcertain cell surface markers characteristic of stem cells and/or theabsence of certain cell surface markers characteristic of differentiatedcells. The methods can also rely on functional assays to measure theengraftment or differentiation potential of the population of cells.Such markers and functional assays are known in the art.

An example of a enriched for stem cells is a population of cellsselected for the CD34⁺Thy⁻1⁺LIN⁻ phenotype as described in U.S. Pat. No.5,061,620. A population of this phenotype typically has an average CAFCfrequency of approximately 1/20 (Murray et al. (1995) supra; Lansdorp etal., J. Exp. Med. 177:1331 (1993)). Methods for isolating highlyenriched populations of hematopoietic stem cells are further provided inU.S. Pat. No. 5,681,559.

As described herein, hematopoietic stem cells have the ability todifferentiate into any of several types of blood cells, including redblood cells, white blood cells, including lymphoid cells and myeloidcells. As described herein, HSCs include hematopoietic cells havinglong-term engrafting potential in vivo. Long term engrafting potential(e.g., long term hematopoietic stem cells) can be determined usinganimal models or in vitro models.

The cells can be enriched for stem cells or immature cells, e.g. in ablood cell lineage, prior to culturing according to the methods providedherein. Cells populations highly enriched in stem cells and methods forobtaining them are described in WO 95/05843; WO 95/03693 and WO95/08105. In a some embodiments, the one or more cells comprise apopulation of cells that is substantially enriched in hematopoietic stemcells. In other embodiments, the cells cultured according to the methodsprovided herein are substantially free of stromal cells.

In some embodiments, the cells used in the methods provided herein areselected or enriched for the presence of or absence of particularmarkers on the surface of the cell. For example, in some embodiments,the cells are selected for the presence of stem cell markers particularfor the animal source of the primary tissue. In other embodiments, thecells are selected for the absence of lineage specific markers. In someembodiments, the cells are selected from the presence of particularmarkers and the absence of other markers. Methods for isolating cellsthat have particular markers or that do not have particular markers arewell known to those skilled in the art.

Regarding lineage specific markers, the absence or low expression oflineage specific markers can be identified by the lack of binding ofantibodies specific to the lineage specific markers. The cells or sourceof cells for use in the methods provided herein can be subjected tonegative selection techniques to remove those cells that express lineagespecific markers and retain those cells which are lineage negative(“Lin⁻”). Lin⁻ generally refers to cells which lack markers such asthose associated with T cells (such as CD2, 3, 4 and 8), B cells (suchas B220, CD48, CD10, 19 and 20), myeloid cells (such as Mac-1, Gr-1,CD14, 15, 16 and 33), natural killer (“NK”) cells (such as CD244, CD2,16 and 56), RBC (such as Ter119, and glycophorin A), megakaryocytes(CD41), mast cells, eosinophils or basophils. Methods of negativeselection are known in the art. Lineage specific markers also includeCD38, HLA-DR and CD71.

Various techniques can be employed to separate the cells by initiallyremoving cells of dedicated lineage or having a particular phenotype.Procedures for separation can include, but are not limited to, physicalseparation, magnetic separation (using antibody-coated magnetic beads),affinity chromatography, cytotoxic agents joined to a monoclonalantibody or used in conjunction with a monoclonal antibody, including,but not limited to, complement and cytotoxins, and “panning” withantibody attached to a solid matrix, e.g., plate, elutriation or anyother convenient technique. Techniques providing accurate and rapidseparation include, but are not limited to, flow cytometry (e.g.,fluorescence activated cell sorting) and cytospin.

The use of physical separation techniques include, but are not limitedto, those based on differences in physical (density gradientcentrifugation and counter-flow centrifugal elutriation), cell surface(lectin and antibody affinity), and vital staining properties(mitochondria-binding dye rho123 and DNA-binding dye Hoechst 33342).These procedures are well known to those of skill in this art.

The cells obtained either with or without enrichment for hematopoieticstems cells as described above can be used immediately or frozen atliquid nitrogen temperatures and stored. The frozen cells can be thawedand used in the methods described herein.

Cell Culture

In some embodiments of expanding HSC cells, the cells, obtained, forexample from a primary tissue source or from a suitable animal, areincubated in a suitable medium. Suitable conditions comprise incubatingat 33° to 39° C., and preferably around 37° C. HSCs can be cultured inan oxygen concentration of 1 to 10%. In some embodiments, the HSCs arecultured under hypoxic conditions. In some embodiments, the cells areincubated under normoxic conditions Normoxic conditions can be, forexample, 5% CO₂ and oxygen at 15% or more. In some embodiments normoxicconditions are 21% O₂. Hypoxic conditions can be, for example, 5% CO₂and 5% O₂.

Media can be replaced throughout the culture period. In anotherembodiment, half of the medium is replaced twice per week with freshmedia. The cells can be cultured from 3 to 30 days. In anotherembodiment, the population of cells including HSCs is cultured for atleast four weeks. In another embodiment, the population of cellsincluding HSCs is cultured for up to two weeks. In another embodiment,the population of cells including HSCs is cultured for 7 to 14 days. Inanother embodiment, the population of cells including HSCs is culturedfor 10 days.

HSCs can be propagated by culturing or incubating one or more cells inan expansion container and in a volume of a suitable medium. The cellscan be cultured such that the culture well contains about 1-100 cellsper well. Where the population of cells is bone marrow, the cells can becultured at a density of about 1×10² cells to about 1×10⁷ cells/mL ofmedium. In another embodiment, the cells can be cultured at a density ofabout 1×10⁵ cells to about 1×10⁶ cells/mL of medium. In anotherembodiment, the population of cells comprises Side Population (SP) bonemarrow cells. The SP bone marrow cells can be cultured at lower density,for example from about 1×10² to 5×10³ cells/ml. In a separate aspect,the population of cells can be derived from mobilized peripheral blood.The mobilized peripheral blood cells can be cultured at a density ofabout 20,000 cells/mL to about 50,000 cells/mL; in another embodiment,the mobilized peripheral blood cells is cultured at a density of about50,000 cells/mL.

Any suitable expansion container, flask, or appropriate tube such as a12, 24 or 96 well plate, 12.5 cm² T flask or gas-permeable bag can beused in the methods provided herein. Such culture containers arecommercially available from Falcon, Corning or Costar. As used herein,“expansion container” also is intended to include any chamber orcontainer for expanding cells whether or not free standing orincorporated into an expansion apparatus.

Hematopoietic stem cells that have been expanded in vitro as describedherein are also provided. It is understood that the descendants of stemcells grown in culture may not be completely identical (eithermorphologically, genetically, or phenotypically) to the parent cell.However, as provided herein, the descendants of the stem cells possessat least some ability to differentiate into one or more blood cell typesas described, supra. Functional characteristics, such as the ability todevelop into one or more blood cell types can be measured, for example,using methods and lineage markers as described herein.

Uses for Ex Vivo Expanded Hematopoietic Stem Cells

The expanded cultured hematopoietic stem cells of the technology can beused for a variety of applications, including transplantation, drugdiscovery, gene cloning, gene delivery, and, gene expression.

Transplantation

The hematopoietic stem cells provided herein can be administered to asubject or an individual. In some embodiments, the hematopoietic stemcells produced by the methods provided herein are used in cell-basedtherapies, such a bone marrow transplantation. Suitable subject orindividuals include any animal as described above. The subject orindividual can be any animal suitable for studying hematopoiesis orcell-based therapies in vivo. vertebrate. The subject or individual canbe any animal in need of cell-based therapy. In some embodiments, theindividual is a mammal. Mammals include, but are not limited to, humans,non-human primates, mice, cows, horses, dogs, cats and the like. In apreferred embodiment, the mammal is a human.

The transplanted stem cells can be autologous (derived from theindividual being treated), allogenic (derived from a donor of the samespecies), or obtained from a histocompatibly matched donor. In someembodiments, the transplanted stem cells can be xenogenic (derived froma animal of a different species from the recipient). Human autologousand allogeneic bone marrow transplantations are currently used astherapies for diseases such as leukemia, lymphoma, and otherlife-threatening diseases.

With respect to administering the expanded cells provided herein to apatient, an effective amount of expanded cells may range from as few asseveral hundred or fewer to as many as several million or more. It willbe appreciated that the number of expanded cells to be administered willvary depending on the specifics of the disorder to be treated, includingbut not limited to size or total volume to be treated, as well as theneeds and condition of the recipient, among other factors familiar tothe medical professional. In some embodiments, between 10³ and 10¹⁰cells per 100 kg person are administered or transplanted into thesubject or individual. Methods of administering or transplanting arewell known in the art and include, for example, infusion. Expanded cellsprovided herein can be administered, for example, by intravenousinfusion.

In some embodiments, a single administration of cells is provided. Inother embodiments, multiple administrations are used. Multipleadministrations can be provided over periodic time periods such as aninitial treatment regime of 3 to 7 consecutive days, and then repeatedat other times.

The expanded cells can be used for reconstituting the full range ofhematopoietic cells in an individual following therapies such as, butnot limited to, radiation treatment and chemotherapy. Such therapiesdestroy hematopoietic cells either intentionally or as a side-effect ofbone marrow transplantation or the treatment of lymphomas, leukemias andother neoplastic conditions, e.g., breast cancer.

Expanded cells provided herein are also useful as a source of cells forspecific hematopoietic lineages. The maturation, proliferation anddifferentiation of expanded hematopoietic cells into one or moreselected lineages may be effected through culturing the cells withappropriate factors including, but not limited to, erythropoietin (EPO),colony stimulating factors, e.g., GM-CSF, G-CSF, or M-CSF, SCF, Flt-3ligand, interleukins, e.g., IL-1, -2, -3, -4, -5, -6, -7, -8, -13, etc.,or with stromal cells or other cells which secrete factors responsiblefor stem cell regeneration, commitment, and differentiation.

Drug Discovery

Hematopoietic stem cells provided by the methods described herein areuseful for drug discovery. For example, culture conditions or growthfactors that promote or inhibit such biological responses of stem cellscan be identified by exposing the cells to the conditions or factors tobe tested. In this way one may also identify, for example, receptors forthese factors or agents that interfere with the biological activity ofthe factor.

The hematopoietic stem cells produced by the methods provided herein canbe used in assays for differentiating stem cells into varioushematopoietic lineages. These assays may be readily adapted in order toidentify substances such as factors which, for example, promote orinhibit stem cell self-regeneration, commitment, or differentiation.

Gene Cloning Strategies

The hematopoietic cells provided herein can be used to identify andclone genes whose expression is associated with proliferation,commitment, differentiation, and maturation of stem cells or otherhematopoietic cells, e.g., by subtractive hybridization or by expressioncloning using monoclonal antibodies specific for target antigensassociated with these biological events or characteristic of ahematopoietic cell type.

Gene Delivery and Expression

Hematopoietic stem cells are also important targets for gene deliveryand expression in a subject. Accordingly the hematopoietic cellsprovided herein can be genetically altered prior to reintroducing thecells into an individual. For example a gene whose expression isexpected to have a therapeutic effect on the individual can beintroduced into one more of the hematopoietic cells provided herein. Thecells can be genetically altered before or after being cultured and/orexpanded as described herein. Methods for introducing genes into thecultured cells are well known in the art.

In some aspects of the technology, individuals can be treated bysupplementing, augmenting and/or replacing defective and/or damagedcells with cells that express a therapeutic gene. The cells may bederived from cells of a normal matched donor or stem cells from theindividual to be treated (i.e., autologous). By introducing normal genesin expressible form, individuals suffering from such a deficiency can beprovided the means to compensate for genetic defects and eliminate,alleviate or reduce some or all of the symptoms.

Expression vectors may be introduced into and expressed in autologous orallogeneic expanded hematopoietic cells, or the genome of cells may bemodified by homologous or non-homologous recombination by methods knownin the art. In this way, one may correct genetic defects in anindividual or provide genetic capabilities naturally lacking in stemcells. For example, diseases including, but not limited to,β-thalassemia, sickle cell anemia, adenosine deaminase deficiency,recombinase deficiency, and recombinase regulatory gene deficiency maybe corrected in this fashion. Diseases not associated with hematopoieticcells may also be treated, e.g., diseases related to the lack ofsecreted proteins including, but not limited to hormones, enzymes, andgrowth factors. Inducible expression of a gene of interest under thecontrol of an appropriate regulatory initiation region will allowproduction (and secretion) of the protein in a fashion similar to thatin the cell which normally produces the protein in nature.

Transduction of Hematopoietic Stem Cell Cultures

The hematopoietic stem cells provided herein can be geneticallymodified. The introduction of the gene into the hematopoietic stem cellcan be by standard techniques, e.g. infection, transfection,transduction or transformation. The HSC cells can be transduced with atherapeutic gene. For example, the transduction can be via a viralvector such as a retroviral vector (e.g. as described in for example, WO94/29438, WO 97/21824 and WO 97/21825) or a pox viral vector. Whentransduction is ex vivo, the transduced cells are subsequentlyadministered to the recipient. Thus, the technology provided hereinencompasses treatment of diseases amenable to gene transfer into HSCs,by administering the gene ex vivo or in vivo by the methods disclosedherein. For example, diseases including, but not limited to,β-thalassemia, sickle cell anemia, adenosine deaminase deficiency,recombinase deficiency, recombinase regulatory gene deficiency, etc. canbe corrected by introduction of a therapeutic gene. Other indications ofgene therapy are introduction of drug resistance genes to enable normalstem cells to have an advantage and be subject to selective pressureduring chemotherapy. Suitable drug resistance genes include, but are notlimited to, the gene encoding the multidrug resistance (MDR) protein.

Examples of modes of gene transfer include e.g., naked DNA, CaPO₄precipitation, DEAE dextran, electroporation, protoplast fusion,lipofection, cell microinjection, and viral vectors, adjuvant-assistedDNA, gene gun, catheters, etc. In another embodiment, a viral vector isused.

One or more polynucleotide of interest can be inserted into a vectorusing methods well known in the art. For example, insert and vector DNAcan be contacted, under suitable conditions, with a restriction enzymeto create complementary ends on each molecule that can pair with eachother and be joined together with a ligase. Alternatively, syntheticnucleic acid linkers can be ligated to the termini of restrictedpolynucleotide. These synthetic linkers contain nucleic acid sequencesthat correspond to a particular restriction site in the vector DNA.Additionally, an oligonucleotide containing a termination codon and anappropriate restriction site can be ligated for insertion into a vectorcontaining, for example, some or all of the following: a selectablemarker gene, such as the neomycin gene for selection of stable ortransient transfectants in mammalian cells; enhancer/promoter sequencesfrom the immediate early gene of human CMV for high levels oftranscription; transcription termination and RNA processing signals fromSV40 for mRNA stability; SV40 polyoma origins of replication and ColE1for proper episomal replication; versatile multiple cloning sites; andT7 and SP6 RNA promoters for in vitro transcription of sense andantisense RNA. Other means are well known and available in the art.

Modification of hematopoietic stem cells can comprise the use of anexpression cassette created for either constitutive or inducibleexpression of the introduced transgene. Such an expression cassette caninclude regulatory elements such as a promoter, an initiation codon, astop codon, and a polyadenylation signal. Suitable elements that areoperable in the stem cells or in cells that arise from the stem cellsafter infusion into an individual can be used. Moreover, it is necessarythat these elements be operably linked to the nucleotide sequence thatencodes the protein such that the nucleotide sequence can be expressedin the stem cells and thus the protein can be produced. Initiationcodons and stop codons are generally considered to be part of anucleotide sequence that encodes the protein.

Examples of promoters that may be used to cause expression of theintroduced sequence in specific cell types include Granzyme A forexpression in T-cells and NK cells, the CD34 promoter for expression instem and progenitor cells, the CD8 promoter for expression in cytotoxicT-cells, and the CD11b promoter for expression in myeloid cells. Inaddition, regulatable promoters can be used. Regulatable promoters suchas inducible promoters are available commercially.

The exogenous genetic material that includes the transgene operablylinked to the regulatory elements may remain present in the cell as afunctioning cytoplasmic molecule, a functioning episomal molecule or itmay integrate into the cell's chromosomal DNA. Exogenous geneticmaterial may be introduced into cells where it remains as separategenetic material in the form of a plasmid. Alternatively, linear DNA,which can integrate into the chromosome, may be introduced into thecell. When introducing DNA into the cell, reagents, which promote DNAintegration into chromosomes, may be added. DNA sequences, which areuseful to promote integration, may also be included in the DNA molecule.Alternatively, RNA may be introduced into the cell.

Selectable markers can be used to monitor uptake of the desired geneinto the hematopoietic stem cells of the technology. These marker genescan be under the control of any promoter or an inducible promoter. Theseare well known in the art and include genes that change the sensitivityof a cell to a stimulus such as a nutrient, an antibiotic, etc. Genesinclude those for neo, puro, and tk, multiple drug resistance (MDR),etc. Other genes express proteins that can readily be screened for suchas green fluorescent protein (GFP), blue fluorescent protein (BFP),luciferase, and LacZ.

As used herein, therapeutic gene can be an entire gene or only thefunctionally active fragment of the gene capable of compensating for thedeficiency in the patient that arises from the defective endogenousgene. Therapeutic gene also encompasses antisense oligonucleotides orgenes useful for antisense suppression and ribozymes forribozyme-mediated therapy. Therapeutic genes that encode dominantinhibitory oligonucleotides and peptides as well as genes that encoderegulatory proteins and oligonucleotides also are encompassed by thistechnology. Generally, gene therapy will involve the transfer of asingle therapeutic gene although more than one gene may be necessary forthe treatment of particular diseases. The therapeutic gene can be anormal, e.g., wild-type, copy of the defective gene or a functionalhomolog. In a separate embodiment, the therapeutic gene is a dominantinhibiting mutant of the wild-type. More than one gene can beadministered per vector or alternatively, more than one gene can bedelivered using several compatible vectors. Depending on the geneticdefect, the therapeutic gene can include the regulatory and untranslatedsequences. For gene therapy in human patients, the therapeutic gene willgenerally be of human origin although genes from other closely relatedspecies that exhibit high homology and biologically identical orequivalent function in humans may be used, if the gene product does notinduce an adverse immune reaction in the recipient. For example, aprimate insulin gene whose gene product is capable of converting glucoseto glycogen in humans would be considered a functional equivalent of thehuman gene. The therapeutic gene suitable for use in treatment will varywith the disease. For example, a suitable therapeutic gene for treatingsickle cell anemia is a normal copy of the globin gene. A suitabletherapeutic gene for treating SCID is the normal ADA gene.

EXAMPLES Results Culture of Total Human Cord Blood Cells in the Presenceof Angptl5 or Angptl3 Stimulates Ex Vivo Expansion of Human HSCs.

Total human cord blood cells were cultured in STIF medium containingAngptl3 or Angptl5. (Conklin, D. et al. Genomics 62, 477-82 (1999);Zeng, L. et al. J Hum Genet. 48, 159-62 (2003)).

2.5×10⁷ total cord blood cells were seeded at a density of 1×10⁶cells/ml serum-free STIF medium containing 100 ng/ml Angptl3 or Angptl5,and total cell numbers were counted at indicated time. After 23 days ofculture, the number of total cells in the presence of Angptl3 increasedabout 10 fold to 2.6±0.3×10⁸ (FIG. 1A. diamonds), and the cells culturedwith Angptl5 increased to 2.2±0.3×10⁸ (squares). The cultured cellscontained mostly suspension cells with a minor adherent subpopulation.

NOD/SCID repopulation assays were conducted to test whether exvivo-expanded cells were capable of engraftment. FIG. 1B shows theamount of human chimerism in the bone marrow of NOD/SCID micetransplanted with 1×10⁶ uncultured human mononuclear cord blood cells,or the cultured progeny of 1×10⁶ initial human cord blood cells. Eachsymbol represents the engraftment of a single transplanted mouse assayedat two months post-transplant (n=5−12). (* Significantly different fromlane 1 value. Student's t-test, p<0.001.) Thus, 1×10⁶ or 3×10⁶uncultured cells, or the cultured progenies of 1×10⁶ initial cells wereinjected into sublethally irradiated NOD/SCID recipients. When 9.2×10⁶cells, cultured with Angptl5 for 19 days (which is the progeny of 1×10⁶initially plated total cord blood cells), were transplanted, an averagehuman hematopoietic chimerism of 8.8% was observed 2 months aftertransplantation (FIG. 1B, lane 2). This is much greater than the 0.6%engraftment shown by the equivalent 1×10⁶ uncultured cells (FIG. 1B,lane 1; p<0.001, student's t-test). The cells cultured in the presenceof Angptl3 for 23 days also engrafted recipients, with an averagechimerism of 7.1% (FIG. 1B, lane 3).

Non-Transfected 293T Cells Stimulates Ex Vivo Expansion of HSCs

Surprisingly, as demonstrated herein, serum-free conditioned mediumcollected from non-transfected 293T cells stimulates ex vivo expansionof HSCs

Twenty freshly isolated CD45.2 bone marrow SP Sca-1+ CD45+ cells werecultured for 10 days in serum-free IMDM supplemented with 10 ng/ml SCF,20 ng/ml TPO, 20 ng/ml IGF-2, and 10 ng/ml FGF-1 (STIF medium; FIG. 2A,bar 1), in freshly collected serum-free conditioned STIF medium from293T cells (bar 2), or in the same conditioned medium after freeze/thaw(bar 3). The cultured cells were co-transplanted with 1×10⁵ CD45.1 totalbone marrow cells into CD45.1 recipients (n=5-6).

FIG. 2B shows the multilineage contribution of cultured cells atconditions represented by bar 2 of FIG. 2A at 5 months post-transplant(n=6). Data shown in the top panel are representative FACS plots ofperipheral blood mononuclear cells from one mouse at 5 monthspost-transplant (from bar 2 of FIG. 1A). Percentages of cells in eachquadrant are listed. The summary of percent repopulation data from micein bar 2 of FIG. 1A for T-lymphoid, B-lymphoid, and myeloid cells isplotted in the bottom panel.

Serum-free 293T conditioned medium was analyzed by mass spectrometry inorder to identify potential candidate proteins that stimulated ex vivoexpansion of HSCs. Peptides from several proteins were identified. Apartial list of peptides identified in the mass spectrometry analysis ofthe fraction of serum-free IMDM based conditioned medium of 293T cellsthat contained proteins smaller than 70 kD is shown in Table I. Proteinsfound in common with the control serum-free IMDM sample are not shown.

TABLE I Accession No. P18965 PO1033 POCOP6 P35555 P36955 Gene IGFBP-2Timp-1 NPS Fibrillin-1 PEDF # peptides 37 20 12 13 15 Total 46 22 24 2222 peptides % of 8.45% 5.32% 15.01% 5.70% 2.99 coverage M.W. 35114 2315610096 26129 46313

As demonstrated herein, IGFBP-2 is expressed in serum-free 293Tconditioned medium. FIG. 3 shows western blot analysis of purified humanIGFBP-2 (positive control; lane 1), serum-free 3T3 conditioned medium(negative control; lane 2), and serum-free 293T conditioned medium (lane3) detected by anti-human IGFBP-2 polyclonal antibody.

Purified IGFBP-2 Stimulates Ex Vivo Expansion of HSCs.

As demonstrated herein, IGFBP-2 stimulates ex vivo expansion of HSCs.Twenty CD45.2 bone marrow SP Sca-1⁺CD45⁺ cells were cultured for 5 daysin STIF medium with 100 ng/ml Angptl3 (bars 1 and 4); in the same mediumwith 500 ng/ml IGFBP-2 (bars 2 and 5); and in the same medium with 200ng/ml Timp-1 (bars 3 and 6) (see FIG. 4A). The cells were thencotransplanted with 1×10⁵ CD45.1 total bone marrow cells into CD45.1recipients (n=5). Engraftments at 1 month or 4 months post-transplantare shown in FIG. 4A. (* Significantly different from bars 4 and 6values. Student's t-test, p<0.05.)

Twenty CD45.2 bone marrow SP Sca-1⁺ CD45⁺ cells were cultured for 10days in serum-free medium with 10 ng/ml SCF, 20 ng/ml TPO, 10 ng/mlFGF-1 (STF medium), and 100 ng/ml Angptl3 (bars 1 and 4); in STF mediumwith 500 ng/ml IGFBP-2 (bars 2 and 5); and in STF medium with 500 ng/mlIGFBP-2 and 100 ng/ml Angptl3 (bars 3 and 6) (see FIG. 4B). The cellswere then cotransplanted with 1×10⁵ CD45.1 total bone marrow cells intoCD45.1 recipients (n=6-7). Engraftments at 1 month or 4 monthspost-transplant are shown in FIG. 4B. (* and ** Significantly differentfrom bar 4 or bar 5 value respectively. Student's t-test, p<0.05.)

FIG. 4C shows limiting dilution analysis of the repopulating ability ofadult BM SP CD45⁺Sca-1⁺ cells before culture (left) and after culturefor 21 days in serum-free conditioned STF medium containing 100 ng/ml ofpurified Angptl3 and 500 ng/ml IGFBP-2 (right). Irradiated CD45.1congenic mice were injected with 1×10⁵ CD45.1 BM competitor cells and 1,5, 25, or 100 freshly isolated SP CD45⁺Sca-1⁺ cells (left; n=24) or thecultured progenies of 0.2, 1, 4, or 10 SP CD45+Sca-1+ cells (right;n=26). 100 Freshly isolated SP Sca-1+CD45+ cells and the culturedprogeny of 4 or 10 input cells repopulated all recipients and these datapoints are not plotted. Plotted is the percentage of recipient micecontaining less than 1% CD45.2 populations in nucleated peripheral bloodcells 4 months after transplant versus the number of cells injected.

Culture of Human Cord Blood CD133⁺ Cells in the Presence of Angptl5 andIGFBP-2 Stimulates Ex Vivo Expansion of HSCs by Over 250 Fold.

It was surprisingly found that IGFBP-2 can replace IGF-2 in supportingthe ex vivo expansion of mouse HSCs. In addition, in the presence of STFmedium, IGFBP-2 promoted the ex vivo expansion human cord blood HSCsCD133⁺ cells by over 250 fold.

Culture of 1×10⁵ human cord blood CD133⁺ cells was initiated inserum-free STF medium, or in serum-free STF medium supplemented with 500ng/ml Angptl5 and 500 ng/ml IGFBP-2 and cultured in a low O₂ environment(5% O₂). Total cell numbers were counted. As shown in FIG. 5A, thenumber of total cells increased greater than 200 fold after 11 days ofculture either in serum-free STF medium or serum-free STF mediumcontaining Angptl5 and IGFBP-2.

IGFBP-2 and Angptl5 Promote Engraftment and Chimerism of Human CordBlood Cells in a Mouse Model

The amount of human chimerism in the bone marrow of NOD/SCID micetransplanted with 8,000 or 15,000 uncultured (fresh) human cord blood CD133⁺ cells, or the progeny from 8,000 initial CD133⁺ cells cultured inserum-free STF medium with or without Angptl5 and IGFBP-2 for 11 dayswas measured. 8,000 uncultured CD133⁺ cells were capable of engraftmentin 1 out of 7 recipients 2 months post-transplant, and the averagechimerism was 0.2% (FIG. 5B, lane 1). 15,000 uncultured CD133⁺ cellsshowed an increased but still modest engraftment; there was positiveengraftment of 4 of 8 mice (average chimerism 2.0% of total cells; FIG.5B, lane 2). In striking contrast, 2.1×10⁶ cells after culture withserum free STF medium with Angptl5 and IGFBP-2, that is, the progeny of8,000 initial cells after 11 days of culture, engrafted all recipientmice, and showed significantly increased chimerism relative to 8,000 or15,000 uncultured sells (average 39.5%) (FIG. 5B, lane 4; p<0.05,student's t-test). The cultured progeny of the same number of initialcells (8,000) cultured in STF medium without Angptl5 and IGFBP-2 (now1.6×10⁶ cells) only exhibited a poor engraftment, similar to that oftheir uncultured counterparts (FIG. 5B, lane 3). Thus, Angptl5 andIGFBP-2 support the ex vivo expansion of human SCID-repopulating cells(SRCs) in a defined medium. Each symbol represents the engraftment of asingle transplanted mouse assayed at two months post-transplant (n=7−8).(* Significantly different from lanes 1-3 values. Student's t-test,p<0.05.)

FIG. 5C shows human hematopoietic engraftment at 2 months in arepresentative mouse that was transplanted with uncultured (fresh) orcultured human cord blood CD133⁺ cells. Representative FACS plots ofbone marrow cells from one mouse at the condition represented by lane 1of (FIG. 5B) (Fresh), or at the condition represented by lane 4 of (FIG.5B) (Cultured), at 2 months post-transplant. Percentages of cells ineach quadrant are listed. Transplant with cells cultured in serum-freeSTF medium containing Angptl5 and IGFBP-2 (lane 4 of FIG. 5B) displayeda much higher engraftment of total hematopoietic (CD45/71⁺), myeloid(CD15/66b⁺), B-lymphoid (CD34⁻CD19/20⁺), and primitive (CD34⁺) humancells than the mouse transplanted with uncultured cells (lane 1 of FIG.5B).

The summary of multi-lineage engraftment of mice transplanted withuncultured cells (FIG. 5B lane 2) and cells cultured in STF mediumcontaining Angptl5 and IGFBP-2 (FIG. 5B lane 4) is shown in FIG. 5D. Theprogeny of 8,000 cells, after culture, repopulated myeloid and lymphoidlineages 2 months post-transplant, demonstrating the expansion of humanstem cell activity. Some mice transplanted with uncultured cells hadzero percent donor repopulation and these data points are not plotted.(* Values are significantly different from the values of the unculturedcells. Student's t-test, p<0.05.) FIG. 5D shows human hematopoieticengraftment at 2 months in a representative mouse that was transplantedwith uncultured or cultured human cord blood CD133⁺ cells. The progenyof 8,000 cells, after culture, repopulated myeloid and lymphoid lineages2 months post-transplant, demonstrating the expansion of human stem cellactivity.

Expansion of Human HSCs in Culture

To measure the self-renewal potential of SCID-repopulating cells (SRCs),bone marrow was collected from the primary mice transplanted withuncultured cells (lane 2 of FIG. 5B) or cells cultured in STF mediumcontaining Angptl5 and IGFBP-2 (lane 4 of FIG. 5B) and transplanted theminto sublethally irradiated secondary recipients. Bone marrow aspiratefrom one hind leg from a primary recipient was used to transplant twosecondary recipients. Multilineage engraftment in secondary NOD/SCIDrecipients was assayed at 5-8 weeks post-transplant (n=12 micetransplanted). While uncultured cells could not engraft secondaryrecipients (not shown), the cultured cells, again, showed positiveengraftment after secondary transplantation (FIG. 5E). These dataindicate a net expansion of human HSCs during the initial cultureperiod. Two additional independent experiments demonstrated that humanHSCs were dramatically expanded in culture using the methods describedherein.

Limiting Dilution Analysis of Human Cord Blood CD133⁺ Cells Transplantedinto NOD/SCID Mice after Culture at Normal or Low Oxygen Levels.

The culture system provided herein dramatically expands human SRCs asdemonstrated by several additional independent experiments. In onerepresentative experiment, 2×10⁵ human cord blood CD133⁺ cells werecultured in STF medium containing 500 ng/ml Angptl5 and 100 ng/mlIGFBP-2 under normal or low O₂ conditions. The numbers of total cells(FIG. 6A) and CD34⁺ cells (FIG. 6B) were counted. After 10 days ofculture, the number of total cells in these two conditions did notdiffer significantly and both increased greater than 200 fold (FIG. 6A).Nevertheless, a higher number of CD34⁺ primitive cells was observedafter 5 days of culture at normal O₂ versus low O² (FIG. 6B).

Limiting dilution assays were performed to quantitate the SRCfrequencies before (FIG. 6C) and after (FIG. 6D) culture. Asdemonstrated in FIGS. 6B and 6C, after a 10-day culture, the number ofSRCs cultured in serum-free STF medium containing Angptl5 and IGFBP-2 atlow or normal O₂ increased by 8- or 20-fold, respectively. Plotted isthe percentage of recipient mice containing less than 1% humanhematopoietic populations in recipient mouse bone marrow 6-8 weeks aftertransplant versus the number of input or input-equivalent cellsinjected. The progeny of 10,000 input cells cultured at normal or low O₂repopulated all recipients and these data points (zero percent negativemice) are not plotted. The frequency of repopulating cells (CRU) forthis particular sample of uncultured CD133⁺ cells is 1 per 64,075 cells(95% confidence interval for mean: 1/23,919 to 1/171,643, n=25). Thatis, as calculated from Poisson statistics, injection of on average of64,075 uncultured human CD133⁺ cells is sufficient to repopulate 63%(=1-1/e) of transplanted mice. When cells were cultured in STF mediumcontaining Angptl5 and IGFBP-2 at low O₂, the CRU frequency was 1/7,814input equivalent cells (95% confidence interval for mean: 1/3,432 to1/17,791, n=26), ˜8 fold greater than that of the uncultured cells.Strikingly, when the cells were cultured at normal O₂, the CRU frequencyincreased to 1/3,209 input equivalent cells (95% confidence interval formean: 1/1,889 to 1/5,453, n=27). This indicates that the total number offunctional SRCs increased ˜20 fold.

FIG. 6E shows multilineage engraftment in NOD/SCID recipientstransplanted with 20,000 uncultured CD133⁺ cells (left panel, n=8) orcultured progeny from 5,000 initial CD133⁺ cells at normal O₂ (rightpanel, n=10). Some mice transplanted with uncultured cells had zeropercent donor repopulation and these data points are not plotted. (totalhematopoietic (cols. 1, and 5, CD45/71+), myeloid (cols. 2 and 6,CD15/66b+), B− lymphoid (cols. 3 and 7, CD34−CD19/20+), and primitive(cols. 4 and 8 CD34+) lineages are shown. (* Value is significantlydifferent from the value of the uncultured cells. Student's t-test,p<0.05.) As demonstrated in FIG. 6E, these cultured cells had muchgreater levels of multi-lineage engraftment than uncultured cells.

It has been suggested that hypoxia improves expansion of human SRCs.(Danet, et al., J Clin Invest 112, 126-35 (2003)). As demonstratedherein, profound expansion of SRCs was observed in a hypoxia condition.Unexpectedly, the SRC expansion was even greater under normoxicpressure.

Human cord blood CD34⁺ cells cultured in the presence of Angptl5 andIGFBP-2 also achieved expansion of SRCs (data not shown).

The use of Angptls and IGFBP-2 for the ex vivo expansion of human HSCsallows increased clinical use of cord blood for bone marrowtransplantation because human HSCs can now be expanded ex vivo in adefined medium. The technology provided herein will be useful for thedevelopment of novel strategies of cell and gene therapies that utilizeHSCs.

Angiopoietin 2 Competitive Reconstitution Analysis

Twenty CD45.2 donor cells were cultured for 10 d in serum-freeconditioned STIF medium or in the same medium with 500 ng/ml purifiedhuman angiopoietin 1, human angiopoietin 2, mouse angiopoietin 3, orhuman angiopoietin 4. The cells cotransplanted with 1×10⁵ freshlyisolated CD45.1 competitor bone marrow cells, into recipient mice. Themixture injected intravenously via the retro-orbital route into each ofa group of 6-9 week old CD45.1 mice previously irradiated with a totaldose of 10 Gy. Reconstitution 4 months post transplant was measured. Tomeasure reconstitution of transplanted mice, peripheral blood wascollected at the indicated times post-transplant and the presence ofCD45.1⁺ and CD45.2⁺ cells in lymphoid and myeloid compartments weremeasured as described (Zhang, C. C. and Lodish, H. F. Blood 105, 4314-20(2005)). Briefly, peripheral blood cells were collected by retro-orbitalbleeding, followed by lysis of red blood cells and staining withanti-CD45.2-FITC, and anti-CD45.1-PE, or anti-Thy1.2-PE (for T-lymphoidlineage), anti-B220-PE (for B-lymphoid lineage), anti-Mac-1-PE,anti-Gr-1-PE (cells costaining with anti-Mac-1 and anti-Gr-1 were deemedthe myeloid lineage), or anti-Ter119-PE (for erythroid lineage)monoclonal antibodies (BD Pharmingen). As shown in the FIG. 7,angiopoietin 2 stimulates ex vivo expansion of HSCs.

Methods

Mice.

C57 BL/6 CD45.2 and CD45.1 mice were purchased from the JacksonLaboratory or the National Cancer Institute. NOD/SCID(NOD.CB17-Prkdcscid/J) mice were purchased from the Jackson Laboratoryand were maintained at the Whitehead Institute animal facility. Allanimal experiments were performed with the approval of M.I.T. Committeeon Animal Care.

Culture Medium.

Serum-free STIF medium is StemSpan serum-free medium (StemCellTechnologies) supplemented with 10 μg/ml heparin (Sigma), 10 ng/ml mouseSCF, 20 ng/ml mouse TPO, 20 ng/ml mouse IGF-2 (all from R&D Systems),and 10 ng/ml human FGF-1 (Invitrogen). Serum-free STF medium is the samemedium without IGF-2. Indicated amounts of purified Angptl3 (a gift fromR&D Systems), Angptl5 (Abnova, Taiwan), or IGFBP-2 (R&D Systems) wereadded. Conditioned medium was collected from confluent 293T or 3T3 cellsafter overnight culture.

Mouse HSC Culture.

Twenty BM SP Sca-1⁺ CD45⁺ cells isolated from 8-10 week old C57BL/6CD45.2 mice were plated in one well of a U-bottom 96-well plate (3799;Corning) with 160 μl of indicated medium. Cells were cultured at 37° C.in 5% CO₂ and normal O₂. For the purpose of competitive transplantation,cells were pooled from at least 6 culture wells and mixed withcompetitors before the indicated numbers of cells were transplanted intoeach mouse.

Human Cell Culture.

Human total cord blood mononuclear cells were purchased from Cambrex.Cells were plated at 1×10⁶ cells/ml of STIF medium, with 100 ng/mlAngptl3 or Angptl5. Medium volume was increased by adding fresh mediumat day 5, 8, 12, 15, and 18 to maintain cell densities at 5×10⁵-1.5×10⁶cells/ml. Cells were cultured at 37° C. in 5% CO₂ and normal O₂. Humancyropreserved cord blood CD133⁺ cells used in the experiments of FIGS. 5and 6 were purchased from Cambrex and StemCell Technologies Inc. Cellswere plated at 1×10⁴ cells/well in one well of a U-bottom 96-well plate(3799; Corning) with 200 μl of the indicated medium for 2 days. At day3, cells were pooled from individual wells and transferred to 6-wellplates at 5×10⁴ cells/ml. Fresh medium was added at days 4 and 7 to keepthe cell density at 2×10⁵ cells/ml (day 4) or 7×10⁵/ml (day 7). Cellswere cultured at 37° C. in 5% CO₂, and normal O₂ or 5% O₂ (low O₂)levels.

NOD/SCID Transplant.

Uncultured or cultured progeny of human total cord blood mononuclearcells or CD133⁺ cells at indicated days were collected and injectedintravenously via the retro-orbital route into sub-lethally irradiated(350 rad) NOD/SCID mice. Six to eight weeks or at indicated time aftertransplantation, bone marrow nucleated cells from transplanted animalswere analyzed by flow cytometry for the presence of human cells. Forsecondary transplantations, bone marrow aspirates from one hind leg of aprimary recipient were used to transplant two secondary recipients, asdescribed. (Hogan, et al., Proc Natl Acad Sci USA 99, 413-8 (2002)).Calculation of CRUs in limiting dilution experiments was conducted usingL-Calc software (StemCell Technologies). (Zhang, et al.,. Proc Natl AcadSci USA 103, 2184-9 (2006)). Mice were considered to be positive forhuman HSC engraftment when at least 1% (for primary transplantation) or0.1% (for secondary transplantation) CD45/71⁺ human cells were detectedamong the mouse bone marrow cells.

Flow Cytometry.

Donor bone marrow cells were isolated from 8-10 week old C57BL/6 CD45.2mice. SP Sca-1⁺ CD45⁺ cells were isolated as described Zhang, C. C. etal., Nat Med 12, 240-5 (2006). For analyzing repopulation of mouse HSCs,peripheral blood cells of recipient CD45.1 mice were collected byretro-orbital bleeding, followed by lysis of red blood cells andstaining with anti-CD45.2-FITC, and anti-CD45.1-PE, or anti-Thy1.2-PE(for T-lymphoid lineage), anti-B220-PE (for B-lymphoid lineage),anti-Mac-1-PE, anti-Gr-1-PE (cells costaining with anti-Mac-1 andanti-Gr-1 were deemed the myeloid lineage), or anti-Ter119-PE (forerythroid lineage) monoclonal antibodies (BD Pharmingen). The “Percentrepopulation” shown in all Figures except FIG. 1B was based on thestaining results of anti-CD45.2-FITC and anti-CD45.1-PE. In all casesFACS analysis of the above listed lineages was also performed to confirmmultilineage reconstitution.

For analyzing human hematopoietic engraftment in NOD/SCID mice, apublished protocol was followed. (Cashman, et al., J Exp Med 196, 1141-9(2002)). Briefly, bone marrow cells from recipient NOD/SCID mice werestained with anti-human CD45-PE, CD71-PE, CD15-FITC, and CD66b-FITC toquantify the total human hematopoietic (CD45/71⁺) cell population aswell as the subset of exclusively granulopoietic (CD 15/66b⁺) cellswithin this population. Cells were stained with anti-human CD34-FITC andanti-human CD19-PE and CD20-PE to quantify human progenitor (CD34⁺) andB-lineage (CD34⁻CD19/20⁺) populations. In the experiment of FIG. 1, onlytotal human hematopoietic (CD45/71⁺) engraftment was measured.Anti-human CD34-FITC was used to quantitate CD34⁺ cells in culture. Allanti-human antibodies were purchased from Becton Dickinson.

FACS Sorting.

Donor bone marrow cells were isolated from 8-10 week old C57BL/6 CD45.2mice. To sort SP Sca-1⁺CD45⁺ cells, adult mouse bone marrow SP cells(stained as previously described (Zhang, C. C. and Lodish, H. F., Blood103, 2513-21 (2004); Zhang, C. C. and Lodish, H. F., Blood 105, 4314-20(2005)) were further stained with anti-Sca-1-PE and anti-CD45-FITCfollowed by cell sorting on a MoFlo® sorter.

Competitive Reconstitution Analysis.

The indicated numbers of mouse CD45.2 donor cells were mixed with 1×10⁵freshly isolated CD45.1 competitor bone marrow cells, and the mixtureinjected intravenously via the retro-orbital route into each of a groupof 6-9 week old CD45.1 mice previously irradiated with a total dose of10 Gy. To measure reconstitution of transplanted mice, peripheral bloodwas collected at the indicated times post-transplant and the presence ofCD45.1⁺ and CD45.2⁺ cells in lymphoid and myeloid compartments weremeasured as described (Zhang, C. C. and Lodish, H. F. Blood 103, 2513-21(2004), Zhang, C. C. and Lodish, H. F., Blood (2005)). Calculation ofCRUs in limiting dilution experiments was conducted using L-Calcsoftware (StemCell Technologies) (Zhang, et al., Proc Natl Acad Sci USA103, 2184-9 (2006)).

Western Blots.

Purified proteins or crude proteins in conditioned medium were analyzedby electrophoresis on 4-12% NuPage Bis-Tris polyacrylamide gels(Invitrogen), and proteins were electroblotted onto nitrocellulosemembranes. The membranes were probed with anti-human IGFBP-2 polyclonalantibody (AF674, R&D Systems) at 0.1 μg/ml, followed with thehorseradish peroxidase-conjugated donkey-anti-goat antibody and detectedby a chemiluminescence kit (Millipore).

While the technology has been particularly shown and described withreference to specific embodiments, it should be understood by thoseskilled in the art that various changes in form and detail may be madetherein without departing from the spirit and scope of the technology asdefined by the appended claims.

1-25. (canceled)
 26. A method of promoting the expansion ofhematopoietic stem cells in culture, comprising culturing cells in aculture medium comprising angiopoietin 2 under conditions sufficient forexpansion of the cells, wherein at least one of the cells is ahematopoietic stem cell.
 27. The method of claim 26, wherein theangiopoietin 2 is a human angiopoietin
 2. 28. The method of claim 26,wherein the cells are cultured for ten days.
 29. The method of claim 26,wherein the culture medium is serum free medium.
 30. The method of claim29, wherein the culture medium further comprises at least one additionalfactor selected from the group consisting of insulin growth factor(IGF), fibroblast growth factor (FGF), thrombopoietin (TPO), and stemcell factor (SCF).
 31. The method of claim 26, wherein the cells areCD45+, Sca-1+ bone marrow cells.
 32. The method of claim 26, wherein thecells are side population cells.
 33. A culture medium suitable forexpanding hematopoietic stem cells in vitro comprising a serum freemedium and angiopoietin
 2. 34. The culture medium of claim 33, whereinthe angiopoietin 2 is human angiopoietin
 2. 35. The culture medium ofclaim 33, wherein the culture medium further comprises at least oneadditional factor selected from the group consisting of insulin growthfactor (IGF), fibroblast growth factor (FGF), thrombopoietin (TPO), andstem cell factor (SCF).
 36. The culture medium of claim 33, comprisingat least two additional factors selected from the group consisting ofinsulin growth factor (IGF), fibroblast growth factor (FGF),thrombopoietin (TPO), and stem cell factor (SCF).
 37. The culture mediumof claim 35, wherein the IGF is IGF-2.
 38. The culture medium of claim35, wherein the FGF is FGF-1.
 39. The culture medium of claim 35,further comprising IGF-2, FGF-1, SCF and TPO.
 40. The method of claim30, further comprising IGF-2, FGF-1, SCF and TPO.
 41. The method ofclaim 26, wherein the angiopoietin 2 has the amino acid sequence of SEQID NO: 1 or an amino acid sequence having at least 60% sequence identitythereto.
 42. The culture medium of claim 33, wherein the angiopoietin 2has the amino acid sequence of SEQ ID NO: 1 or an amino acid sequencehaving at least 60% sequence identity thereto.
 43. A kit for in vitroexpansion of hematopoietic stem cells comprising a) angiopoietin 2, andb) instructions for expanding hematopoietic stem cells in vitro.
 44. Thekit of claim 43, wherein the angiopoietin 2 is human angiopoietin
 2. 45.The kit of claim 43, wherein the angiopoietin 2 has the amino acidsequence of SEQ ID NO: 1 or an amino acid sequence having at least 60%sequence identity thereto.